Ovarian Ascorbic Acid Depletion Research Articles

Assessment of the biopotency of follitropin alfa and lutropin alfa combined in one injection: a comparative trial in Sprague-Dawley rats

BackgroundThe current study was designed to determine if follitropin alfa (recombinant human follicle-stimulating hormone; r-hFSH) and lutropin alfa (recombinant human luteinizing hormone; r-hLH) biopotencies were unchanged by reconstituting in sterile water for injection and mixing prior to injection.MethodsThe biopotencies of r-hFSH and r-hLH were determined following injection of female Sprague-Dawley rats with a mixture of follitropin alfa revised formulation female (RFF) and lutropin alfa (1:1, r-hFSH:r-hLH). Biopotencies of follitropin alfa RFF and lutropin alfa were measured using ovarian weight and ascorbic acid depletion assays, respectively, and compared with a reference standard. Stock mixtures of follitropin alfa RFF and lutropin alfa (1:1) were prepared within 1 h prior to each respective assay's injection and stored at 6 +/- 2°C. Separate low dose (follitropin alfa RFF 1.5 IU/rat, lutropin alfa 2 IU/rat) and high dose (follitropin alfa RFF 3 IU/rat, lutropin alfa 8 IU/rat) treatments were prepared from stock mixtures or individual solutions by diluting with 0.22% bovine serum albumin saline solution and injected within 1 h of preparation. The main outcome measures were ovarian weight and ovarian ascorbic acid depletion.ResultsFSH bioactivities were similar (p > 0.10) between the individual follitropin alfa RFF test solution (84.2 IU) and follitropin alfa RFF/lutropin alfa (87.6 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2°C. LH bioactivities were similar (p > 0.10) between lutropin alfa (94.7 IU) test solution and lutropin alfa/follitropin alfa RFF (85.3 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2°C for not more than 1 h prior to injection.ConclusionMixing follitropin alfa RFF and lutropin alfa did not alter the bioactivity of either FSH or LH.

Is an immunoassay available for the measurement of bioactive LH in serum?

An in vitro bioassay for luteinizing hormone (LH) is in our opinion the "gold standard" bioassay. The rodent interstitial cell testosterone assay (RICT) is specific for bioactive LH and very sensitive, accurate, and reproducible. Diverse LH standards consistently display parallel dose-response characteristics. Sera also manifest parallel dose-response characteristics throughout reproductive life, with the exception of basal samples from prepubertal children. This indicates that all known hormones with LH bioactivity have a similar bioactive site. The in vivo bioassays for LH used for calibration of World Health Organization standards are more cumbersome and less precise and accurate than the in vitro bioassay. The ovarian ascorbic acid depletion assay corresponds better than the seminal vesicle weight assay with in vitro bioassay. Variation in the ratio of bioactive to immunoreactive LH (B/I) principally reflects variation in LH immunoassay dose-response characteristics, rather than a change in the bioactive moiety of LH. The varying B/I ratio is due to molecular heterogeneity at multiple levels. Different LH standards contain different proportions of nonbioactive but immunoreactive material. The immunoreactive LH isoforms in serum contain different proportions of bioactive material and the isoform distribution differs with reproductive status. Furthermore, the antibodies comprising the various immunoassay systems detect heterogeneous epitopes on LH, which are not necessarily bioactive. B/I ratio disparities indicate lack of specificity of immunoassays for bioactive LH. Polyclonal antibody-based radioimmunoassay requires the use of purified reagents, including a bioactive tracer, in order to achieve high specificity for bioactive LH. The new generation of monoclonal antibody-based immunometric assays yields results that are lower than, but correlate with, LH measured by the in vitro bioassay. The purest of standards, even a recombinant standard, yields results that differ up to 50% or more from one immunoassay to another. Serum LH levels also differ up to two-fold among assays. The immunometric assays have the advantage of being more sensitive and more specific for low levels of LH in serum than radioimmunoassays, but B/I ratio discrepancies remain great. An immunoassay specific for the bioactive "docking site" of human LH isoforms is still needed.

Purification of human pituitary LH and thyrotrophin by hydrophobic chromatography

A new preparative procedure is described for the efficient purification of LH and TSH from frozen human pituitary glands. LH and TSH were isolated simultaneously from crude preparations by hydrophobic-interaction chromatography followed by separation and purification by reverse-phase high-performance liquid chromatography in a single step. Highly purified hormones were prepared in good yields (45 mg LH and 20 mg TSH/1000 glands) and with high biological activities; the potencies of purified LH and TSH were 5.8 x NIH-LH-S1 equivalent in an ovarian ascorbic acid depletion assay and 7.1 U/mg against human TSH MRC Research Standard (T1 70/9) in the McKenzie assay respectively. Cross-contamination by other glycoprotein hormones was low.

Interaction of Human Growth Hormone and Gonadotrophins on the Function of Rat Ovaries

Hypophysectomized and normal female rats were used for studying the interaction of human growth hormone and gonadotrophins in stimulating the rat ovary. Groups of rats were injected with increasing doses of gonadotrophins (Pergonal or Metrodin) in addition to human growth hormone (Norditropin) or placebo. In the test for follicle stimulating hormone activity the weight of the ovaries was recorded. In the hypophysectomized rats Norditropin and Pergonal or Metrodin had a synergistic stimulating effect on the ovary, resulting in a greater number and a larger follicle size. Norditropin had a slight stimulatory effect of its own. In the ovarian ascorbic acid depletion test for luteinising hormone activity Norditropin had a significant effect of its own. In combination with Pergonal an additive effect was seen. The effect of Norditropin was observed in hypophysectomized rats only.

Biopotency of highly purified porcine FSH and human LH on gonadal function

The gonadotrophin preparations that have been used previously to study different aspects of testis function are of limited purity. We have, therefore, purified existing gonadotrophin preparations further. The demonstration of their purity and biological activity are reported as well as their suitability for in-vivo use. After separation of the subunits of the intact hormones by high-performance liquid chromatography followed by analytical sodium dodecylsulphate-polyacrylamide gel electrophoresis, no contaminating proteins could be detected in either the FSH or LH preparation. After immunoadsorption, contamination by other pituitary hormones (TSH, prolactin, FSH, LH and GH) was found to be less than 0.002% by weight for all the hormones tested. The biological activity of porcine FSH (pFSH) measured in a Steelman-Pohley assay was 150-170 times more potent than the NIH-FSH-P1 reference preparation. The biopotency of human LH (hLH) in the ovarian ascorbic acid depletion test was measured and appeared to be 8100-8300 IU/mg against the 68/40 International Standard. The dose-dependent effects of pFSH and hLH on testis weight and the number of FSH and LH receptors were measured in immature (22-day-old) hypophysectomized rats treated for 7 consecutive days. Treatment with FSH induced a dose-dependent increase in testis weight (threefold) when compared with the control. The concentration and total number of LH receptors were increased (two- to sixfold) in a dose-dependent manner. The number of FSH receptors per testis increased while the number of FSH receptors per mg of protein remained unchanged. Administration of human LH to immature hypophysectomized rats had no effect on either LH or FSH receptors, regardless of the dose administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary extract of the ricefield eel Monopterus albus (synbranchidae, teleostei) exhibits gonadotropic activity in the classes mammalia, aves, reptilia and amphibia

1. 1. Pituitary extract of the ricefield eel Monopterus albus demonstrated gonadotropic activity in mammals and non-mammalian vertebrates. 2. 2. Using the rat as the recipient, FSH activity was detected in Monopterus pituitaries in the HCG augmentation test and LH activity in the ovarian ascorbic acid depletion test. 3. 3. Cyclic AMP level in superovulated ovaries, ovarian lactate production and glucose uptake in vitro, plasma testosterone level in males, testicular enzymes, ventral prostate weight and other androgen-dependent parameters were stimulated after treatment with Monopterus pituitary extract. 4. 4. Testicular and ovarian 32p 5+ uptake in the chick, testicular weight in the grass turtle Chinemys reevesi, and ovulation in the amphibians Xenopus laevis and Rana tigrina were enhanced. 5. 5. Both the FSH-like and LH-like activities in Monopterus pituitaries were sensitive to proteolytic enzymes and chemicals that attack the disulfide linkage, carbohydrate moiety, tyrosine, tryptophan and histidine residues. 6. 6. This constitutes the first report of dual gonadotropic activities elicited by a teleost pituitary extract in the mammal in vivo.

Purification and characterization of guinea-pig chorionic gonadotrophin.

A human chorionic gonadotrophin-like protein (GF-1, 1.0 g) from the placentae of 50 guinea-pigs killed at Day 26 of gestation was purified by pH and ammonium salt fractionation followed by column chromatography on DEAE-Sephadex and filtration on Sephadex G-100. Relative to the Second International hCG standard (MRC 61/6) GF-1 had an immunological potency of 21 000 i.u./mg as measured in a specific hCG-beta radioimmunoassay and, using the ovarian ascorbic acid depletion assay, an apparent biological potency of 24 064 i.u./mg. Isoelectric focusing yielded 6 bands between pH 4.4 and 5.7 and the material comprised two non-covalently linked subunits. The Stokes' radii were 3.40 nm for the native preparation, and 2.38 nm and 3.15 nm for GF-1-alpha and GF-1-beta subunits respectively. The guinea-pig placenta therefore produces a chorionic gonadotrophin which on purification has physicochemical, biological and immunological properties similar to those of hCG.

A pitfall in the ovarian ascorbic acid depletion assay (OAAD): effect of unilateral ovariectomy, hCG and asialo-hCG.

The estimation of the effect of desialylation on the biological activity of human chorionic gonadotrophin (hCG), as determined in the ovarian ascorbic acid depletion assay (OAAD), according to the modification in which the animals are used twice, appeared to be dependent on the assay conditions. Animals which received hCG in the first assay (OAAD-1) gave higher potency estimates for hCG and asialo-hCG in OAAD-1. The results of the experiments indicate that the ovarian sensitivity for hCG and asialo-hCG is increased after administration of hCG and unilateral ovariectomy and decreased after administration of asialo-hCG.

OVARIAN BINDING AND INTRINSIC BIOLOGICAL ACTIVITY OF DESIALYLATED HUMAN CHORIONIC GONADOTROPHIN (ASIALO-hCG) IN IMMATURE SUPEROVULATED RATS

The present study was undertaken to investigate the effect of desialylation on the intrinsic in vivo biological activity of hCG. To block the hepatic uptake of asialo-hCG we investigated asialo-orosomucoid. The biological activity of hCG and asialo-hCG was estimated with the ovarian ascorbic acid depletion assay (OAAD). The uptake studies were performed with [125I]hCG and [125I]asialo-hCG. Desialylation of hCG increased its hepatic uptake, almost completely abolished its ovarian uptake, and reduced its in vivo biological activity to less than 1%. Administration of asialo-orosomucoid caused a reduction of the hepatic uptake of asialo-hCG to the level observed for hCG. The ovarian uptake increased, but was lower than observed for hCG, due to a considerably higher renal accumulation of asialo-hCG. The in vivo biological activity of asialo-hCG was approximately 1/5 of the activity of hCG when both were tested in the presence of asialo-orosomucoid. To exclude the interference of renal accumulation, binding and biological activity were also investigated after bilateral nephrectomy. The uptake of radioactivity in the ovaries following injection of [512I]asialo-hCG and asialo-orosomucoid was then almost the same as observed after injection of [125I]hCG alone. When both preparations were now tested in the presence of asialo-orosomucoid, the in vivo biological activity of asialo-hCG appeared to be approximately 1/3 of the value obtained for hCG. It is concluded that the intrinsic in vivo biological activity of hCG is lowered by desialylation.

RABBIT LUTROPIN: PREPARATION, CHARACTERIZATION OF THE HORMONE, ITS SUBUNITS AND RADIOIMMUNOASSAY

The purification of rabbit lutropin is described. A product with a potency of 1.53 X NIH-LH-Sl was obtained as assayed by the ovarian ascorbic acid depletion assay. In a homologous radioimmunoassay, which is described, rabbit lutropin has a potency 4.83 X NIH-LH-Sl. In a radioligand assay, utilizing labeled ovine lutropin as the trace, the relative potency was 0.47 X NIH-LH-Sl measured by 50% inhibition comparison since rabbit lutropin response in this system did not parallel ovine lutropin. A counter-current distribution procedure for separation of rabbit lutropin subunits is described. Amino acid composition of the isolated subunits and intact rabbit lutropin was determined. The carbohydrate composition of the latter is presented; only amino sugar determinations are available for the subunits. The NH2-terminal amino acids are phenylalanine (alpha subunit) and alanine (beta subunit). Preliminary data on COOH-terminal amino acids are provided.

Effect of diverse mammalian and nonmammalian gonadotropins on isolated rat Leydig cells

The isolated rat Leydig cell assay was employed to study species specificity in biological action. Eight mammalian luteinizing hormones (LHs), human chorionic gonadotropin, and pregnant mare serum gonadotropin stimulated testosterone production in this assay with a 250-fold potency range. A high potency correlated with a high carbohydrate content. Comparison of the potencies of these hormones in the Leydig assay, the ovarian ascorbic acid depletion assay, and the Xenopus ovulation assay showed the results of the two rat assays to be the most similar, demonstrating that the receptor as well as the hormone play a role in determining species specificity. The LH specificity of the assay was reexamined and follicle-stimulating hormone (FSH) was found to have a small intrinsic activity. Nonmammalian LHs, which generally possess little or no activity in in vivo mammalian assays, were able to stimulate rat Leydig cells, at 1000x mammalian LH doses. LH specificity was retained with nonmammalian LHs but the potency differences between nonmammalian LHs and FSHs were much less than mammalian LHs and FSHs.

Studies of the binding activity to different gonadal receptors of ovine luteinizing hormone (LH) after chemical modification of lysine residues

Several LH derivatives obtained by modification of the lysine residues were studied by the radioligand receptor assay using either corpora lutea homogenates or testis homogenates and tritiated methylated LH. The binding activity found was very similar to the biological activity as determined in the ovarian ascorbic acid depletion test, although some minor differences were observed. The binding activity of the methylated derivatives was no different from that of native LH, whereas the other alkylated derivatives were less active or inactive. As expected, carbamylated LH was inactive and the activity of guanidinated LH was considerably reduced. Some differences were observed in the binding affinities of these derivatives when comparing corpora lutea with testicular homogenates.

Species specificity in the response of an in vitro amphibian ( Xenopus laevis) ovulation assay to mammalian luteinizing hormones

Relative potencies of pituitary LH and pregnancy gonadotropins (hCG and PMSG) from numerous mammalian species estimated by an in vitro ovulation bioassay in the anuran Xenopus laevis correlated poorly with estimates obtained by various mammalian bioassays. For example, potencies of seven preparations of pituitary LH which varied by about 5-fold in the in vivo mammalian ovarian ascorbic acid depletion (OAAD) bioassay, differed by 140-fold in potency in the anuran bioassay. The ranking of hormonal potencies was also very different in these two assays; e.g., human LH was the most potent in the rat OAAD assay and among the least potent in Xenopus (ovine LH was most potent in Xenopus). Results of the OAAD assay corresponded closely to those observed in several in vitro assays based on steroid production by mammalian (rat and rabbit) tissues. Thus, pronounced species specificity is evident in the response of Xenopus ovaries to gonadotropins from mammalian origin; these findings have important implications for interpreting comparative data on LH purification and biological actions of hormones.

The effect of LH-RH, prostaglandins and synthetic analogues of LH-RH on ovarian metabolism.

Abstract Ovarian ascorbic acid depletion (OAAD) is observed after injection of LH-RH and its analogues in pseudopregnant rats. Injection of prostaglandins is followed by similar changes in ovarian ascorbic acid concentration. A new test system for synthetic LH-RH and its analogues was developed based on OAAD after i.m. or s.c. injection. The potency estimates of this assay were compared with ovulation tests. 16 analogues of LH-RH were tested both in the ovarian ascorbic acid system and in the ovulation assay, with closely correlated potency estimates. The effect of natural prostaglandins was studied in the same test systems. A dose-dependent depletion was found 15 min—1 h after injection of LH-RH or its analogues together with dose-dependent increase in plasma LH and rapid stimulation of progesterone production. In hypophysectomized rats, ovarian ascorbic acid levels were reduced and depletion by LH-RH was abolished. Prostaglandins likewise induced OAAD, but progesterone levels were lowered (PGF 2α ) and no change in plasma LH was observed. Only luteolytic prostaglandins were found effective on OAAD. Ovulation tests in rat primed with PMSG showed dose-dependent ovulation by LH-RH and its analogues. Injection of prostaglandins was ineffective on ovulation and the rate of ovulation induced by LH-RH was not changed by simultaneous prostaglandin treatment. Prostaglandins are inactive with respect to ovulation and decrease progesterone production, whereas LH-RH and its analogues induce both ovulation and progesterone production. Ovarian ascorbic acid depletion in immature rats after LH-RH is a reliable bioassay without requirement for RIA, and may also be a useful assay for luteolytic prostaglandins.

Extraction and purification of a substance with luteinizing hormone releasing activity from the leaves of Avena sativa.

Attempts were made to purify the LH-releasing substance extracted from the leaves of Avena sativa by means of two-step chromatographic procedures using a weakly acidic ion-exchange resin (CG-50,type II) and DEAE-Sephadex A-25 (coarse) with successful results. For preliminary fractionation of such starting materials as dried leaves, fresh leaves, and acetone-extracted powder(crude extracts), 5% acetate-buffered active carbon proved to be more effective than starch zone electrophoresis. From its behavior on chromatography with weakly acidic ion-exchange resins as well as Sephadex gel filtration, the active fraction extracted from the leaves of Avena saliva was assumed to be different from the LH-RH present in the hypothalamus. This partially purified material, however, was demonstrated to have an LH-releasing activity by the ovarian ascorbic acid depletion method using Wistar-Imamichi strain rats. Evidence was presented that its site of action is in the adenohypophysis.

Effect of selective nitration of ovine lutropin on the subunit association and biological activity of the hormone.

Different nitrated lutropin (LH) subunits were tested for their abilities to recombine with their counterpart subunits. (A detailed description of the preparations tested is presented in the preceding paper (Burleigh, B. D., Liu, W.K., and Ward, D.N. (1976) J. Biol. Chem. 251, 308-315).) The results indicated that the fully nitrated beta subunit of ovine LH (oLHBeta) (diatyrosyl oHbeta) can combine with the alpha subunit of ovine LH (oLHalpha) as wen which tyrosine residues alpha21, alpha41, alpha93 are modified) recombined partially with oLHbeta yielding about 40% as recombined product under specified recombination conditions. The fully nitrated oLHalpha (pentaatyrosyl oLH) combined poorly with oLHbeta and only 20 to 25% was obtained as the recombined product. The various nitrated LH and recombined LH derivatives containing separately nitrated subunits were tested for their potency in an in vitro radioligand assay and their in vivo biological potency in the ovarian ascorbic acid depletion assay. In the radioligand assay, the monoatyrosyl oLH and the oLHalpha + diatyrosyl oLHbeta were the most active derivatives and had about the same competitive inhibtion curves as that of oLHalpha + oLHbeta. Diatyrosyl oLH had intermediate native potency and the triatyrosyl oLH, tetraatyrosyl oLH + OLHbeta, and pentaatyrosyl oLHalpha + oLHbeta were the least active derivatives in this series. No tetranitromethane-modified LH derivative in our study was completely devoid of the specific binding activity in the radioligand assay. At a dosage of 1000-fold excess over the 125I-labeled oH, all the preparations tested in this series completely inhibited the specific binding of 125I-labeled oLH to the hormone receptors in rat testis preparations. In the in vivo ovarian ascorbic acid depletion assay, both the monoatyrosyl oLH and oLHalpha + diatyrosyl oLHbeta retained about 50% of its original activity; the diatyrosyl oLH and triatyrosyl oLH had about 10% of the activity and the recombined products of tetraatyrosyl oHalpha + oLHbeta and pentaatyrosyl oLHalpha + oLHbeta showed virtually no activity. Based on the results obtained from this paper and the preceding paper, the role of the individual tyrosine residues in oLH is assessed.

The Role of the Amino Group in Subunit Association and Receptor Site Interaction for Ovine Luteinizing Hormone as Studied by Acylation

Abstract The positively charged amino groups (in physiological pH range) of ovine luteinizing hormone (oLH) and its subunits (oLHα and oLHβ) were acylated to produce negatively charged (maleylated) or neutral groups (carbamylated) in their place. In both reactions, the number of modified e-lysine amino groups in luteinizing hormone (LH), LHα, and LHβ was 10, 9, and 2, respectively. The NH2 termini were also completely acylated in both reactions. Maleylation or carbamylation did not alter the hydrodynamic volume of oLH or oLHβ significantly, although a greater Stokes radius was obtained with the oLHα derivatives compared to native oLHα as judged by Kav values from molecular exclusion chromatography. Maleylated LHβ combined with native LHα as well as the native subunits recombine, but maleylated LHα recombined with native LHβ less effectively. Efficiency of recombination was judged by the quantity of material with a hydrodynamic volume similar to LH under specified recombination conditions. Maleylated LHα and maleylated LHβ recombined very poorly. The series of recombination products with carbamylated subunits were all obtained in yields comparable to those observed with the recombination of the native subunits. Biological activities of the various recombined products were examined by both the in vivo ovarian ascorbic acid depletion assay and the in vitro radioligand receptor assay and the results agreed. Intact oLH which had been carbamylated or maleylated, as well as the recombined subunit derivatives from these reactions were completely inactive. Either the carbamylated or maleylated oLHα plus native oLHβ, and native oLHα plus maleylated oLHβ gave a low biological activity with response characteristics different from that of the native hormone. The recombined native subunits, or native oLHα plus carbamylated oLHβ, gave products that were slightly less potent than native oLH, with response characteristics that paralleled those of the native hormone. These results suggest that the charges on lysine e-amino groups in the oLHα play a role in the recombination with oLHβ, but that the charge on the oLHβ amino groups is not crucial for subunit-subunit interaction. It may also be concluded that the charge on the free amino groups of oLHβ must be positive or neutral for receptor site interaction. At least a portion of the amino groups on the oLHα must carry a positive charge for maximum hormone-receptor site interaction.

Prostaglandin-induced ovarian ascorbic acid depletion.

The object of this study was to determine whether prostaglandins (PG's) have an LH-like action measurable by the ovarian ascorbic acid depletion method. PGE1, E2, or F2α produced a highly significant decrease in the ovarian content of ascorbic acid in intact and hypophysectomized immature rats with luteinized ovaries due to previous treatment with pregnant mares′ serum (PMS) and human chorionic gonadotropin (hCG). Ten min after a single iv injection of PGE1 E2 or F2α in immature rats with luteinized ovaries, serum LH levels were increased significantly as shown by radioimmunoassay, but LH levels in the control groups were undetectable. Ovulation was induced by hCG treatment in 100% of hypophysectomized immature rats treated with PMS, while it was not induced after injection of PGE2 or F2α in similarly treated rats. Histologically, massive luteinization of the ovaries of hCG-treated rats was observed, but the formation of corpora lutea was never observed in the rats treated with PGE2 or F2α. From these observations, we conclude that PG's have a dual effect on corpus luteal function as expressed by ovarian ascorbic acid depletion: an indirect effect by inducing LH secretion, and a direct effect manifested in hypophysectomized rats. The latter is not due to an LH-like action of PG's, because PG's failed to induce ovulation and luteinization in hypophysectomized immature rats pretreated with PMS. (Endocrinology95: 417, 1974)

Assay for the quantitative estimation of the gonadotropin inhibiting material in human urine.

A method for the quantitative estimation of the gonadotropin inhibiting material (GIM) in human urine is standardised using the ovarian ascorbic acid depletion test, the results being expressed in terms of and HCG units. A dose-response relationship with GIM is established. The reliability of the assay is demonstrated. Unlike other methods of assay this method is able to express precisely the quantity of GIM present in the urine. Preliminary data on GIM values in normal subjects as well as patients with endocrine disorders are presented.

The specificity of an ovine pituitary incubation system linked with gonadotrophin assays for detecting gonadotrophin releasing activity.

SUMMARY A sheep pituitary incubation system was developed which may be used to study the release of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as assayed biologically. Specificity was examined by adding at high concentration various materials present in the hypothalamus, a crude acid extract of sheep cerebral cortex or crude or partly purified extracts of ovine hypothalamic tissue. Of the materials tested for LH and FSH releasing activity, oxytocin, adrenaline, synthetic lysine vasopressin and cerebral cortex extract failed to influence LH or FSH release. Natural vasopressin containing unknown proportions of the lysine and arginine forms did not affect FSH release but produced a significant increase in the LH content of the medium, the reason for which was not clear. Noradrenaline failed to influence FSH release but produced an apparent depression in the LH content of the medium due to an action on the LH released or upon the ovarian ascorbic acid depletion assay since the addition of noradrenaline to standard ovine LH reduced the potency in the assay to an almost identical degree. Extracts of ovine hypothalamic tissue consistently increased the LH and FSH content of the medium. The method described may be applicable to the detection and estimation of LH and FSH releasing activity in ovine hypothalamic tissue extracts and to detecting changes in the responsiveness to hypothalamic stimulation of sheep pituitary tissue.